Proteomic Study of the Impact of Hik33 Mutation in Synechocystis sp. PCC 6803 under Normal and Salt Stress Conditions

Abstract Background Mannitol is a C(6) polyol that is used in the food and medical sector as a sweetener and antioxidant, respectively. The sustainable production of mannitol, especially via the direct conversion of CO 2 by photosynthetic cyanobacteria, has become increasingly appealing. However, previous work aiming to achieve mannitol production in the marine Synechococcus sp. PCC 7002 via heterologous expression of mannitol-1-phosphate-5-dehydrogenase ( mtlD ) and mannitol-1-phosphatase ( m1p , in short: a ‘mannitol cassette’), proved to be genetically unstable. Results Here, we explore the stabilizing effect that mannitol production may have on cells faced with osmotic stress, in the freshwater cyanobacterium Synechocystis sp. PCC 6803. We first validated that mannitol can function as a compatible solute in Synechocystis sp. PCC 6803, and in derivative strains in which the ability to produce one or both of the native compatible solutes was impaired. Wild type Synechocystis , complemented with a mannitol cassette, indeed showed increased salt tolerance, which was even more evident in Synechocystis strains in which the ability to synthesize the endogenous compatible solutes was impaired. Next we tested the genetic stability of all these strains with respect to their mannitol productivity, with and without salt stress, during prolonged turbidostat cultivations. The obtained results show that mannitol production under salt stress conditions in the Synechocystis strain that cannot synthesize its endogenous compatible solutes is remarkably stable, while the control strain completely loses this ability in only 6 days. DNA sequencing results of the control groups that lost the ability to synthesize mannitol revealed that multiple types of mutation occurred in the mtlD gene that can explain the disruption of mannitol production. Conclusions Mannitol production in freshwater Synechocsytis sp. PCC6803 confers it with increased salt tolerance. Under this strategy, genetically instability which was the major challenge for mannitol production in cyanobacteria is tackled. This paper marks the first report of utilization of the response to salt stress as a factor that can increase the stability of mannitol production in a cyanobacterial cell factory.

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Using Osmotic Stress to Stabilize Mannitol Production in Synechocystis sp. PCC6803

10.21203/rs.3.rs-17598/v1 ◽

2020 ◽

Author(s):

Wenyang Wu ◽

Wei Du ◽

Ruth Perez Gallego ◽

Klaas J. Hellingwerf ◽

Aniek D. van der Woude ◽

...

Keyword(s):

Salt Stress ◽

Salt Tolerance ◽

Osmotic Stress ◽

Compatible Solutes ◽

Direct Conversion ◽

Cell Factory ◽

Medical Sector ◽

Mannitol Production ◽

Synechocystis Sp ◽

Pcc 6803

Abstract Background Mannitol is a C(6) polyol that is used in the food and medical sector as a sweetener and antioxidant, respectively. The sustainable production of mannitol, especially via the direct conversion of CO 2 by photosynthetic cyanobacteria, has become increasingly appealing. However, previous work aiming to achieve mannitol production in the marine Synechococcus sp. PCC 7002 via heterologous expression of mannitol-1-phosphate-5-dehydrogenase ( mtlD ) and mannitol-1-phosphatase ( m1p , in short: a ‘mannitol cassette’), proved to be genetically unstable. Results Here, we explore the stabilizing effect that mannitol production may have on cells faced with osmotic stress, in the freshwater cyanobacterium Synechocystis sp. PCC 6803. We first validated that mannitol can function as a compatible solute in Synechocystis sp. PCC 6803, and in derivative strains in which the ability to produce one or both of the native compatible solutes was impaired. Wild type Synechocystis , complemented with a mannitol cassette, indeed showed increased salt tolerance, which was even more evident in Synechocystis strains in which the ability to synthesize the endogenous compatible solutes was impaired. Next we tested the genetic stability of all these strains with respect to their mannitol productivity, with and without salt stress, during prolonged turbidostat cultivations. The obtained results show that mannitol production under salt stress conditions in the Synechocystis strain that cannot synthesize its endogenous compatible solutes is remarkably stable, while the control strain completely loses this ability in only 6 days. DNA sequencing results of the control groups that lost the ability to synthesize mannitol revealed that multiple types of mutation occurred in the mtlD gene that can explain the disruption of mannitol production. Conclusions Mannitol production in freshwater Synechocsytis sp. PCC6803 confers it with increased salt tolerance. Under this strategy, genetically instability which was the major challenge for mannitol production in cyanobacteria is tackled. This paper marks the first report of both stable mannitol production directly from CO 2 in cyanobacteria, and of the utilization of the response to salt stress as a factor that can stabilize production in a cyanobacterial cell factory.

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